Recombinant Anti-Histone H3 (tri methyl K9) antibody [EPR16601] - ChIP Grade (ab176916)
Key features and details
- Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
- Rabbit monoclonal [EPR16601] to Histone H3 (tri methyl K9) - ChIP Grade
- Suitable for: CUT&Tag-seq, PepArr, ChIP, ChIP-sequencing, ICC/IF, IHC-P, WB, Dot blot, ChIC/CUT&RUN-seq
- Reacts with: Mouse, Rat, Human
Related conjugates and formulations
Overview
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Product name
Anti-Histone H3 (tri methyl K9) antibody [EPR16601] - ChIP Grade
See all Histone H3 primary antibodies -
Description
Rabbit monoclonal [EPR16601] to Histone H3 (tri methyl K9) - ChIP Grade -
Host species
Rabbit -
Tested applications
Suitable for: CUT&Tag-seq, PepArr, ChIP, ChIP-sequencing, ICC/IF, IHC-P, WB, Dot blot, ChIC/CUT&RUN-seqmore details -
Species reactivity
Reacts with: Mouse, Rat, Human -
Immunogen
Synthetic peptide. This information is proprietary to Abcam and/or its suppliers.
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Positive control
- WB: HeLa and NIH/3T3 whole cell lysates; IHC-P: Human colon, mouse liver and rat liver tissues; ICC/IF: HeLa and NIH/3T3 cells; ChIP: SAT-alpha primer pair ab269263; ChIP-seq: Chromatin from HeLa cells. CUT&Tag seq: Sox10 chr15:79,091,798-79,329,947 (Mouse mm10 genome used)
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General notes
This product is a recombinant monoclonal antibody, which offers several advantages including:
- - High batch-to-batch consistency and reproducibility
- - Improved sensitivity and specificity
- - Long-term security of supply
- - Animal-free production
Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMAb® patents.
Properties
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Form
Liquid -
Storage instructions
Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C long term. Avoid freeze / thaw cycle. -
Storage buffer
Preservative: 0.01% Sodium azide
Constituents: 59% PBS, 40% Glycerol, 0.05% BSA -
Concentration information loading...
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Purity
Protein A purified -
Clonality
Monoclonal -
Clone number
EPR16601 -
Isotype
IgG -
Research areas
Associated products
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Alternative Versions
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Compatible Secondaries
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Isotype control
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Positive Controls
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Recombinant Protein
Applications
The Abpromise guarantee
Our Abpromise guarantee covers the use of ab176916 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Application | Abreviews | Notes |
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CUT&Tag-seq |
1/100.
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PepArr |
Use at an assay dependent concentration.
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ChIP |
Use 2 µg for 25 µg of chromatin.
Use SAT-alpha ChIP primer pair ab269263 as positive control. |
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ChIP-sequencing |
Use 4µg for 107 cells.
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ICC/IF |
1/2000.
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IHC-P |
1/500. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
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WB | (6) |
1/1000. Detects a band of approximately 15 kDa (predicted molecular weight: 15 kDa).
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Dot blot |
1/1000.
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ChIC/CUT&RUN-seq |
Use 2µg for 105 cells.
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Notes |
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CUT&Tag-seq
1/100. |
PepArr
Use at an assay dependent concentration. |
ChIP
Use 2 µg for 25 µg of chromatin. Use SAT-alpha ChIP primer pair ab269263 as positive control. |
ChIP-sequencing
Use 4µg for 107 cells. |
ICC/IF
1/2000. |
IHC-P
1/500. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol. |
WB
1/1000. Detects a band of approximately 15 kDa (predicted molecular weight: 15 kDa). |
Dot blot
1/1000. |
ChIC/CUT&RUN-seq
Use 2µg for 105 cells. |
Target
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Function
Core component of nucleosome. Nucleosomes wrap and compact DNA into chromatin, limiting DNA accessibility to the cellular machineries which require DNA as a template. Histones thereby play a central role in transcription regulation, DNA repair, DNA replication and chromosomal stability. DNA accessibility is regulated via a complex set of post-translational modifications of histones, also called histone code, and nucleosome remodeling. -
Sequence similarities
Belongs to the histone H3 family. -
Developmental stage
Expressed during S phase, then expression strongly decreases as cell division slows down during the process of differentiation. -
Post-translational
modificationsAcetylation is generally linked to gene activation. Acetylation on Lys-10 (H3K9ac) impairs methylation at Arg-9 (H3R8me2s). Acetylation on Lys-19 (H3K18ac) and Lys-24 (H3K24ac) favors methylation at Arg-18 (H3R17me).
Citrullination at Arg-9 (H3R8ci) and/or Arg-18 (H3R17ci) by PADI4 impairs methylation and represses transcription.
Asymmetric dimethylation at Arg-18 (H3R17me2a) by CARM1 is linked to gene activation. Symmetric dimethylation at Arg-9 (H3R8me2s) by PRMT5 is linked to gene repression. Asymmetric dimethylation at Arg-3 (H3R2me2a) by PRMT6 is linked to gene repression and is mutually exclusive with H3 Lys-5 methylation (H3K4me2 and H3K4me3). H3R2me2a is present at the 3' of genes regardless of their transcription state and is enriched on inactive promoters, while it is absent on active promoters.
Methylation at Lys-5 (H3K4me), Lys-37 (H3K36me) and Lys-80 (H3K79me) are linked to gene activation. Methylation at Lys-5 (H3K4me) facilitates subsequent acetylation of H3 and H4. Methylation at Lys-80 (H3K79me) is associated with DNA double-strand break (DSB) responses and is a specific target for TP53BP1. Methylation at Lys-10 (H3K9me) and Lys-28 (H3K27me) are linked to gene repression. Methylation at Lys-10 (H3K9me) is a specific target for HP1 proteins (CBX1, CBX3 and CBX5) and prevents subsequent phosphorylation at Ser-11 (H3S10ph) and acetylation of H3 and H4. Methylation at Lys-5 (H3K4me) and Lys-80 (H3K79me) require preliminary monoubiquitination of H2B at 'Lys-120'. Methylation at Lys-10 (H3K9me) and Lys-28 (H3K27me) are enriched in inactive X chromosome chromatin.
Phosphorylated at Thr-4 (H3T3ph) by GSG2/haspin during prophase and dephosphorylated during anaphase. Phosphorylation at Ser-11 (H3S10ph) by AURKB is crucial for chromosome condensation and cell-cycle progression during mitosis and meiosis. In addition phosphorylation at Ser-11 (H3S10ph) by RPS6KA4 and RPS6KA5 is important during interphase because it enables the transcription of genes following external stimulation, like mitogens, stress, growth factors or UV irradiation and result in the activation of genes, such as c-fos and c-jun. Phosphorylation at Ser-11 (H3S10ph), which is linked to gene activation, prevents methylation at Lys-10 (H3K9me) but facilitates acetylation of H3 and H4. Phosphorylation at Ser-11 (H3S10ph) by AURKB mediates the dissociation of HP1 proteins (CBX1, CBX3 and CBX5) from heterochromatin. Phosphorylation at Ser-11 (H3S10ph) is also an essential regulatory mechanism for neoplastic cell transformation. Phosphorylated at Ser-29 (H3S28ph) by MLTK isoform 1, RPS6KA5 or AURKB during mitosis or upon ultraviolet B irradiation. Phosphorylation at Thr-7 (H3T6ph) by PRKCBB is a specific tag for epigenetic transcriptional activation that prevents demethylation of Lys-5 (H3K4me) by LSD1/KDM1A. At centromeres, specifically phosphorylated at Thr-12 (H3T11ph) from prophase to early anaphase, by DAPK3 and PKN1. Phosphorylation at Thr-12 (H3T11ph) by PKN1 is a specific tag for epigenetic transcriptional activation that promotes demethylation of Lys-10 (H3K9me) by KDM4C/JMJD2C. Phosphorylation at Tyr-42 (H3Y41ph) by JAK2 promotes exclusion of CBX5 (HP1 alpha) from chromatin.
Monoubiquitinated by RAG1 in lymphoid cells, monoubiquitination is required for V(D)J recombination (By similarity). Ubiquitinated by the CUL4-DDB-RBX1 complex in response to ultraviolet irradiation. This may weaken the interaction between histones and DNA and facilitate DNA accessibility to repair proteins. -
Cellular localization
Nucleus. Chromosome. - Information by UniProt
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Database links
- Entrez Gene: 8350 Human
- Entrez Gene: 8351 Human
- Entrez Gene: 8352 Human
- Entrez Gene: 8353 Human
- Entrez Gene: 8354 Human
- Entrez Gene: 8355 Human
- Entrez Gene: 8356 Human
- Entrez Gene: 8357 Human
see all -
Alternative names
- H3 histone family member E pseudogene antibody
- H3 histone family, member A antibody
- H3/A antibody
see all
Images
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Chromatin was prepared from HeLa cells according to the Abcam X-ChIP protocol. Cells were fixed with formaldehyde for 10 minutes. The ChIP was performed with 25µg of chromatin, 2µg of ab176916 (blue), and 20µl of Protein A/G sepharose beads. No antibody was added to the beads control (yellow). The immunoprecipitated DNA was quantified by real time PCR (Taqman approach for active and inactive loci, Sybr green approach for heterochromatic loci). Primers and probes are located in the first kb of the transcribed region.
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ChIC/CUT&RUN sequencing - Anti-Histone H3 (tri methyl K9) antibody [EPR16601] - ChIP Grade (ab176916)
CUT&RUN was performed using the ChIC/CUT&RUN pAG-MNAse ab285373, 105 HeLa cells and 2µg of ab176916 [EPR16601]. The resulting DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 10 million reads. The ChIP data was conducted on chromatin prepared from HeLa cells. Cells were fixed with 1% formaldehyde for 10 minutes. ChIP was performed with 107 HeLa cells and 4 µg of ab176916 [EPR16601]. ChIP DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 30 million reads.
Additional screenshots of mapped reads can be downloaded here.
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CUT&Tag sequencing - Anti-Histone H3 (tri methyl K9) antibody [EPR16601] - ChIP Grade (ab176916)This experiment and image is courtesy of Dr Marek Bartosovic, Gonçalo Castelo-Branco Group, Karolinska Institutet.
CUT&Tag-seq was performed using 200,000 Oli-neu (Oligodendrocyte progenitor) cells. Cells were permeabilized with 0.05% Digitonin and 0.01% NP-40 for 3 minutes. A 1:100 dilution of Recombinant Anti-Histone H3 (tri methyl K9) antibody [EPR16601] - ChIP Grade (ab176916) was used, along with a Guinea pig anti-rabbit Secondary. DNA was seg using Illumina NovaSeq S Prime to a depth of 19 million reads.
This image is courtesy of Dr Marek Bartosovic, Gonçalo Castelo-Branco Group, Karolinska Institutet.
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Chromatin was prepared from HeLa cells. Cells were fixed with 1% formaldehyde for 10 minutes. ChIP was performed with 107 HeLa cells and 4 µg of ab176916 [EPR16601]. ChIP DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 30 million reads.
Additional screenshots of mapped reads can be downloaded here.
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All lanes : Anti-Histone H3 (tri methyl K9) antibody [EPR16601] - ChIP Grade (ab176916) at 1/1000 dilution
Lane 1 : HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate
Lane 2 : NIH/3T3 (Mouse embryonic fibroblast cell line) whole cell lysate
Lysates/proteins at 10 µg per lane.
Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution
Predicted band size: 15 kDa
Observed band size: 15 kDaBlocking/Dilution buffer: 5% NFDM/TBST.
Exposure time: Lane 1: 1 second; Lane 2: 4 seconds.
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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Histone H3 (tri methyl K9) antibody [EPR16601] - ChIP Grade (ab176916)
Immunohistochemical analysis of paraffin-embedded human colon tissue labeling Histone H3 (tri methyl K9) with ab176916 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Nuclear staining on human colon is observed. Counter stained with Hematoxylin.
Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.
Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
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Immunocytochemistry/ Immunofluorescence - Anti-Histone H3 (tri methyl K9) antibody [EPR16601] - ChIP Grade (ab176916)
Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized NIH/3T3 (Mouse embryonic fibroblast cell line) cells labeling Histone H3 (tri methyl K9) with ab176916 at 1/2000 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing nuclear staining on NIH/3T3 cell line. The nuclear counter stain is DAPI (blue).
Tubulin is detected with Anti-alpha Tubulin mouse MAb (ab7291) at 1/1000 dilution, followed by Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) (ab150120) secondary antibody at 1/1000 dilution (red).
The negative controls are as follows:
-ve control 1: ab176916 at 1/2000 dilution followed by Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) (ab150120) secondary antibody at 1/1000 dilution.
-ve control 2: Anti-alpha Tubulin mouse MAb (ab7291) at 1/1000 dilution followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution. -
Dot blot analysis of Histone H3 (tri methyl K9) peptide (Lane 1), Histone H3K9 unmodified peptide (Lane 2), Histone H3 (crotonyl K4) peptide (Lane 3) and Histone H3K4 unmodified peptide (Lane 4) labeled using ab176916 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) secondary antibody at 1/1000 dilution.
Blocking/Dilution buffer: 5% NFDM/TBST.
Exposure time: 3 minutes.
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ab176916 was tested in Peptide array against 501 different modified and unmodified histone peptides; each peptide is printed on the array at six concentrations (each in triplicate).
Circle area represents affinity between the antibody and a peptide: all antigen-containing peptides are displayed as red circles, all other peptides as blue circles. The affinity is calculated as area under curve when antibody binding values are plotted against the corresponding peptide concentration. Each circle area is normalized to the peptide with the strongest affinity.
The complete dataset, including full list of all peptides and information on the position of each peptide in the diagram, can be downloaded here.
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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Histone H3 (tri methyl K9) antibody [EPR16601] - ChIP Grade (ab176916)
Immunohistochemical analysis of paraffin-embedded mouse liver tissue labeling Histone H3 (tri methyl K9) with ab176916 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Nuclear staining on mouse liver is observed. Counter stained with Hematoxylin.
Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.
Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Histone H3 (tri methyl K9) antibody [EPR16601] - ChIP Grade (ab176916)
Immunohistochemical analysis of paraffin-embedded rat liver tissue labeling Histone H3 (tri methyl K9) with ab176916 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Nuclear staining on rat liver is observed. Counter stained with Hematoxylin.
Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.
Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
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Immunocytochemistry/ Immunofluorescence - Anti-Histone H3 (tri methyl K9) antibody [EPR16601] - ChIP Grade (ab176916)
Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (Human epithelial cell line from cervix adenocarcinoma) cells labeling Histone H3 (tri methyl K9) with ab176916 at 1/2000 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing nuclear staining on HeLa cell line. The nuclear counter stain is DAPI (blue).
Tubulin is detected with Anti-alpha Tubulin mouse MAb (ab7291) at 1/1000 dilution, followed by Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) (ab150120) secondary antibody at 1/1000 dilution (red).
The negative controls are as follows:
-ve control 1: ab176916 at 1/2000 dilution followed by Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) (ab150120) secondary antibody at 1/1000 dilution.
-ve control 2: Anti-alpha Tubulin mouse MAb (ab7291) at 1/1000 dilution followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution.
Protocols
Datasheets and documents
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SDS download
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Datasheet download
Certificate of Compliance
References (21)
ab176916 has been referenced in 21 publications.
- Puvvula PK & Moon AM Novel Cell-Penetrating Peptides Derived From Scaffold-Attachment- Factor A Inhibits Cancer Cell Proliferation and Survival. Front Oncol 11:621825 (2021). PubMed: 33859938
- Wan QL et al. N6-methyldeoxyadenine and histone methylation mediate transgenerational survival advantages induced by hormetic heat stress. Sci Adv 7:N/A (2021). PubMed: 33523838
- Guo Y et al. Sirt3-mediated mitophagy regulates AGEs-induced BMSCs senescence and senile osteoporosis. Redox Biol 41:101915 (2021). PubMed: 33662874
- Zhang H et al. MicroRNA-545 suppresses progression of ovarian cancer through mediating PLK1 expression by a direct binding and an indirect regulation involving KDM4B-mediated demethylation. BMC Cancer 21:163 (2021). PubMed: 33588776
- Amiad-Pavlov D et al. Live imaging of chromatin distribution reveals novel principles of nuclear architecture and chromatin compartmentalization. Sci Adv 7:N/A (2021). PubMed: 34078602